Search Images Maps Play YouTube News Gmail Drive More »
Sign in
Screen reader users: click this link for accessible mode. Accessible mode has the same essential features but works better with your reader.

Patents

  1. Advanced Patent Search
Publication numberUS4725677 A
Publication typeGrant
Application numberUS 06/752,178
PCT numberPCT/EP1984/000244
Publication date16 Feb 1988
Filing date10 Aug 1984
Priority date18 Aug 1983
Fee statusPaid
Also published asCA1234060A1, DE3329892A1, DE3474964D1, EP0152459A1, EP0152459B1, WO1985000816A1
Publication number06752178, 752178, PCT/1984/244, PCT/EP/1984/000244, PCT/EP/1984/00244, PCT/EP/84/000244, PCT/EP/84/00244, PCT/EP1984/000244, PCT/EP1984/00244, PCT/EP1984000244, PCT/EP198400244, PCT/EP84/000244, PCT/EP84/00244, PCT/EP84000244, PCT/EP8400244, US 4725677 A, US 4725677A, US-A-4725677, US4725677 A, US4725677A
InventorsHubert Koster, Nanda D. Sinha
Original AssigneeBiosyntech Gmbh
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Process for the preparation of oligonucleotides
US 4725677 A
Abstract
The invention relates to a process for the preparation of oligonucleotides by the following steps: reaction of a nucleoside with a phosphine derivative, reaction of the nucleotide derivative thus obtained with a nucleoside bonded to a polymeric carrier, oxidation of the carrier-bound nucleoside-nucleotide thus obtained with formation of phosphotriester groups, blocking of free primary 5'-OH groups, elimination of a protective group from the terminal 5'-OH group, where appropriate single or multiple repetition of the abovementioned steps to introduce further nucleoside phosphate or oligonucleoside phosphate units, and cleavage of the nucleoside-carrier bond and, where appropriate, elimination of all protective groups present in the oligonucleoside phosphates. The phosphine derivative used is a compound of the general formula III ##STR1## in which X and L can react with OH groups of the sugar units in the oligonucleotides, and R3 is a protective group which can be liberated by β-elimination.
Images(5)
Previous page
Next page
Claims(19)
We claim:
1. A process for the preparation of oligonucleotides of the general formula I
in which B denotes a nucleoside base, R1 denotes hydrogen, hydroxyl or hydroxyl which is protected by a removable protective group and n denotes an integer from 1 to 200, comprising the steps of
(a) reacting a nucleoside of the general formula II ##STR14## in which R1 as defined as above, and R2 denotes a removable protective group and B' denotes the nucleoside base B protected by the protective groups which can be eliminated, with a phosphine derivative of the general formula III ##STR15## in which R3 is a protective group which can be eliminated, and X and L are groups which react with hydroxyl groups in the sugar moieties of the nucleotides or nucleosides, in the presence of a base to thereby form a nucleotide phosphite
(b) reacting the nucleotide phosphite obtained in step (a) and represented by the formula IV: ##STR16## in which B', R1, R2, R3 and L are as defined above, with a nucleoside, of the general formula V, bound to a polymeric carrier ##STR17## in which B' and R1 are as defined above and C denotes the polymeric carrier;
(c) oxidizing the carrier-bound nucleoside-nucleotides obtained in step (b) and represented by the formula: ##STR18## in which B', R1, R2, R3 and C are as defined above, with formation of phosphotriester groups,
(d) blocking free primary 5'-OH groups, which have not been reacted in the reaction according to step (b), with permanent protective groups;
(e) eliminating the protective group R2 ;
(f) optionally repeating steps (a) to (e) to introduce further nucleoside phosphate or oligonucleoside phosphate units; and
(g) cleaving the nucleoside carrier bond and optionally eliminating the protective groups present in the oligonucleoside phosphates,
which process comprises using in step (a) as the phosphine derivative of the general formula III a compound in which R3 denotes a group of the formula VII ##STR19## in which the groups Y, which can be identical or different, represent hydrogen, methyl, and/or ethyl and Z represents an electron-attracting group, where, in the phosphine derivative of the formula III, X is chlorine, bromine, CN or SCN and L is CN or SCN, a secondary amino radical of the formula (VIII)
--NR2 4 
where the groups R4 are primary, secondary, or tertiary alkyl radicals having 1-10 carbon atoms, or together form a cycloalkyl radical having 5-7 carbon atoms, which can contain one or two nitrogen, oxygen, or sulfur atoms as hereoatoms, or are imidazole, triazole, tetrazole, 3-nitro-1,2,4-triazole, thiazole, pyrrole, benzotriazole, benzohydroxytriazole, imidazole substituted in the phenyl moiety, triazole substituted in the phenyl moiety, tetrazole substituted in the phenyl moiety, 3-nitro-1,2,4-triazole substituted in the phenyl moiety, thiazole substituted in the phenyl moiety, pyrrole substituted in the phenyl moiety, benzotriazole substituted in the phenyl moiety, or benzohydroxytrizole substituted in the phenyl moiety.
2. The process as claimed in claim 1, in which is used a phosphine derivative of the formula III in which X is chlorine or bromine, and L is a secondary amino radical of the formula (VIII)
--NR2 4                                           (VIII)
where the groups R4 are primary, secondary or tertiary alkyl radicals having 1-10 carbon atoms, or together form a cycloalkyl radical having 5-7 carbon atoms, which can contain one or two nitrogen, oxygen or sulfur atoms as heteroatoms, or are imidazole, triazole, tetrazole, 3-nitro-1,2,4-triazole, thiazole, pyrrole, benzotriazole, benzohydroxytriazole, imidazole substituted in the phenyl moiety, triazole substituted in the phenyl moiety, tetrazole substituted in the phenyl moiety, 3-nitro-1,2,4-triazole substituted in the phenyl moiety, thiazole substituted in the phenyl moiety, pyrrole substituted in the phenyl moiety, benzotriazole substituted in the phenyl moiety, or benzohydroxytrizole substituted in the phenyl moiety.
3. The process as claimed in claim 1 or 2, in which is used a phosphine derivative of the formula (III) in which X is chlorine, L is an N,N-dimethylamino, -diethylamino or -diisopropylamino group or N-morpholino group, and R3 is a β-cyanoethyl group.
4. A method of preparing oligonucleotides of the general formula: ##STR20## wherein B is a nucleoside base, R1 is hydrogen, hydroxyl or hydroxyl which is protected by removable nucleoside protective groups, and n denotes an integer from 1 to 200, comprising the steps of:
(a) reacting a nucleotide phosphite represented by the formula: ##STR21## wherein B' is a nucleoside base B protected by a base protective group which can be eliminated, R1 is as defined above, R2 is 4,4' dimethoxytrityl or 4,4',4" trimethoxytrityl; and L is N,N-dimethylamino, N,N-diethylamino, N,N-diisopropylamino, or N-morpholino, with a nucleoside bound to a polymeric carrier, of the general formula: ##STR22## wherein B' and R1 are as defined above and C represents the polymeric carrier, to produce a carrier bound nucleoside-nucleotide of the formula: ##STR23## wherein B', R1, R2, R3 and C are as defined above; (b) oxidizing the carrier bound nucleoside-nucleotide;
(c) blocking free primary 5'-OH groups, which have not been reacted in the reaction of step (a), with permanent protective groups;
(d) eliminating the protecting group R2 ;
(f) repeating steps (a) to (d) to introduce further nucleoside phosphate units; and
(g) cleaving the nucleoside-carrier bond and optionally eliminating protective groups present in the oligonucleoside phosphates.
5. A method of synthesizing oligonucleotides, comprising the steps of:
(a) coupling a nucleoside β-cyanoethyl-protected phosphoramidite to a carrier-bound nucleoside to produce a carrier bound nucleoside-nucleotide having a phosphite triester linkage;
(b) oxidizing the phosphite triester to form a phosphate triester;
(c) optionally coupling additional nucleoside β-cyanoethyl-protected phosphoramidites to the carrier bound nucleoside-nucleotide and, after each coupling step, oxidizing the resulting phosphite triester to form a phosphate triester, to form a carrier bound polynucleotide;
(d) removing the β-cyanoethyl protecting groups; and
(e) removing the polynucleotide from the carrier.
6. A method of claim 5, wherein the nucleoside β-cyanoethyl phosphoramidite is a nucleoside β-cyanoethyl N,N-dimethylphosphoramidite, N,N-diethylphosphoramidite, N,N-dipropylphosphoramidite or N,N-morpholino phosphoramidite.
7. A method of claim 6, wherein the carrier is controlled pore glass.
8. A method of claim 7, wherein the β-cyanoethyl protecting group is removed with simultaneous removal of the polynucleotide from the carrier, by concentrated aqueous ammonia.
9. In a method of polynucleotide synthesis, comprising sequentially coupling nucleotide phosphoramidites to produce a polynucleotide, wherein the phosphorus atoms of the nucleotide phosphoramidites are protected by methyl groups, the improvement wherein the phosphorus protecting group are cyanoethyl groups.
10. A method of synthesizing oligonucleotides, comprising the steps of:
a. coupling a nucleoside β-cyanoethyl-protected phosphoramidite to a nucleoside, the nucleoside being bound to a polymeric carrier via an ester bond to produce a carrier-bound nucleoside-nucleotide having a phosphite triester linkage;
b. oxidizing the phosphite triester to form a phosphate triester linkage;
c. sequentially coupling additional nucleoside β-cyanoethyl protected phosphoramidite to the carrier-bound nucleoside-nucleotide, and after each coupling step, oxidizing the resulting phosphite triester linkage to a phosphate triester to produce a carrier-bound polynucleotide;
d. treating the carrier bound polynucleotide with concentrated ammonia to remove the β-cyanoethyl phosphate protecting group and hydrolyzing the ester bond to the carrier to remove the polynucleotide from the carrier.
11. A method of claim 10, wherein the nucleoside β-cyanoethyl phosphoramidite is a nucleoside β-cyanoethyl N,N-dimethylphosphoramidite, N,N-diethylphosphoramidite; N,N-dispropylphosphoramidite or N,N-morpholino phosphoramidite.
12. A method of claim 10, wherein the carrier is controlled pore glass.
13. A protected nucleotide having the formula: ##STR24## where, B' is a nucleoside base protected by a base protective group which can be eliminated;
R1 is H, OH, or a hydroxyl group which is protected by a removable nucleoside protective group;
R2 is a removable protective group;
R3 is ##STR25## L is CN, SCN, or NR2 4 ; R4 is a primary, secondary or tertiary alkyl radical having 1-10 carbon atoms, or R2 4 is a cyloalkyl radical having 5-7 carbon atoms or a cycloalkyl radical having 5-7 atoms comprising atoms and one or two nitrogen, oxygen or sulfur atoms as heteroatoms;
Y is H, CH3, or CH2 CH3 ; and Z is a halgen, CN, NO2, phenyl substituted in the o, o' or p positions with a halogen, CN or NO2 radical, phenylthio, phenylsulfoxy, or phenylsulfonyl, where the phenyl radicals, may be substituted in the o, o' or p positions with a halgen, CN or NO2 radical, or where the group ##STR26## may be replaced by CF3, CCl3, or CBr3.
14. A protected nucleotide as in claim 13, wherein Z is CN.
15. A protected nucleotide as in claim 14, wherein R3 is CH2 --CH2 --CN.
16. A protected nucleotide as in claim 13, wherein R2 is 4,4'-dimethoxytrityl or 4,4"-trimethoxytrityl.
17. A protected nucleotide having the formula: ##STR27## where, R1 is H, OH, or a hydroxyl group which is protected by a removable nucleoside protective group;
R2 is 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl;
B' is a nucleoside base protected by a base protective group which can be eliminated
R3 is CH2 --CH2 --CN; and
L is N,N-dimethylamino, N,N-diethylamino, N,N-diisopropylamino or N-morpholino.
18. A protected nucleotide having the formula: ##STR28## where, R1 is H, OH, or a hydroxyl group which is protected by a protective group selected from the group consisting of trityl groups, acyl groups and silyl ether groups;
R2 is 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl;
B' is a nucleoside base selected from the group consisting of adenine, guanine, cytosine, thymine, uracil and analogs thereof which are protected by acyl groups or Schiff bases;
R3 is CH2 --CH2 --CN; and
L is N,N-dimethylamino, N,N-diethylamino, N,N-diisopropylamino, or N-morpholino.
19. A protected nucleotide of claim 18, wherein R1 is H and B' is adenine, guanine, cytosine, thymine or uracil wherein the adenine or guanine is protected by a benzoyl group and the guanine is protected by an isobutyl group.
Description
DESCRIPTION

The invention relates to a process for the preparation of oligonucleotides of the general formula I indicated in claim 1. The oligonucleotides prepared according to the invention have defined sequences and can be used as specific primers and probes and are of great importance for the synthesis of complete genes (Arzneimittelforschung 30, 3a, 548, (1980)).

According to the most recent state of the art, oligonucleotides are prepared by either the phosphate or phosphite triester method using polymeric carriers (Nachr. Chem. Tech. Lab. 29, 230 (1981)). In order to be able to construct defined sequences, it is necessary for the individual units (nucleosides or nucleotides) to be provided with suitable protective groups. In this context, base-labile acyl groups are generally used for the protection of the exocyclic amino groups on the heterocyclic nucleobases, and a base-labile ester bond is used to attach the oligonucleotide chain to the polymeric carrier in a customary manner, and acid-labile trityl ether groups are used to protect the primary 5'-OH group. The phosphate protective group used in the phosphate triester method is customarily either the 2-chlorophenyl or the 4-chlorophenyl group, with an ester-type bond, which can only be removed by attack of a base or a nucleophile on the phosphorus atom. This type of step is inherently undesirable since it involves the risk of cleavage of the internucleotide phosphate ester bond. This risk has been greatly reduced by the use of oximate anions (Tetrahedron Lett. 19, 2727 (1978)), although these also attack the phosphorus atom in an undesired manner in the crucial step and, moreover, have the disadvantage that a relatively small amount of desired oligonucleotide is contaminated with every large amounts of involatile salts which are difficult to extract. This not only makes the working up and subsequent purification of the synthesized oligonucleotide difficult but also leads to considerable material losses.

In the phosphite triester method, the methyl group with an ester-type bond is customarily used as the phosphate protective group which can be removed by attack of a nucleophile on the methyl C atom (J. Amer. Chem. Soc. 99, 3526 (1977)). Since attack on the P atom is avoided, there is likewise avoidance of the risk of cleavage of the internucleotide bond. The nucleophile customarily used is thiophenol/triethylamine, which are unpleasant to manipulate and, moreover, lead to involatile compounds which are difficult to extract and which, as mentioned above, both make work-up difficult and lead to considerable material losses.

Although the actual synthesis of oligonucleotides by the solid phase/phosphite or phosphate triester method takes place very efficiently and rapidly, the preparation of oligonucleotides of defined sequence remains very time-consuming. This is primarily due to the problems of the subsequent work-up and purification which take up a multiple of the actual synthesis time. The process of the invention operates at this point and provides in this connection a crucial technical improvement.

In order to obtain compounds of the formula I indicated in claim 1, ##STR2## in which B denotes a nucleobase, for example adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U) or their analogs, and R1 denotes hydrogen, hydroxyl or hydroxyl which is protected by the protective groups customary in nucleotide chemistry, and n denotes an integer from 1 to 200, according to the invention a variety of defined reaction steps are carried out, as follows:

(a) Reaction of a nucleoside of the general formula II. ##STR3##

R1 of the general formula II can be hydrogen; in this case the compounds of the formula I are oligodeoxynucleotides. The group R1 can also be hydroxyl or hydroxyl which is, where appropriate, protected by the protective groups customary in nucleotide chemistry. Examples of protective groups of this type are trityl, monomethoxytrityl and dimethoxytrityl, acyl, for example acetyl, benzoyl; tetrahydropyranyl, methoxytetrahydropyranyl, o-nitrobenzyl and silyl ethers, such as, for example, t-butyldiphenylsilyl ethers. A general review of the protective groups customary in nucleotide chemistry is to be found in, for example, Tetrahedron 1981, pages 363-369, Liebigs Ann. Chem. 1978, 839-850, and Nucleic Acids Research, Symposium Series No. 7, 1980, 39-59.

R2 is likewise a protective group customary in nucleotide chemistry according to the abovementioned publications, preferably the acid-labile 4,4'-dimethoxytrityl or 4,4',4"-trimethoxytrityl group. B' can likewise have a protective group customary in nucleotide chemistry according to the abovementioned prior publications.

The nucleoside of the formula II is reacted according to the invention with a phosphine derivative of the general formula III according to claim 1. ##STR4##

In the general formula, X denotes chlorine, bromine, CN or SCN; L denotes chlorine, bromine, CN, SCN or an amino radical of the formula --NR2 4 (formula VIII), where the groups R4 denote primary, or secondary or tertiary alkyl radicals having 1-10 carbon atoms, or together denote a cycloalkyl radical having 5-7 carbon atoms, optionally with alkyl branches, and/or can contain one or two nitrogen, oxygen and/or sulfur atoms as heteroatoms. The group L can also form a reactive heterocyclic radical, the imidazolyl, triazolyl, tetrazolyl, 3-nitro-1,2,4-triazolyl, thiazolyl, pyrrolyl, benzotriazolyl (optionally with substituents in the phenyl moiety) or benzohydroxytriazolyl (optionally with substituents in the phenyl ring) and the like.

R3 in the phosphine derivative of the general formula (III) is, according to the invention, a group of the general formula VII, ##STR5## which can be removed with the aid of bases by β-elimination and in which Y denotes hydrogen, methyl or ethyl. Z represents an electron-attracting group, for example, halogen, such as fluorine, chlorine or bromine, CN or NO2. Z can also denote phenyl, phenylthio, phenylsulfoxy or phenylsulfonyl, it being possible for the phenyl radicals to be substituted in the o, o'-position and/or p-position with halogen, CN or NO2. It is also possible for one of the groups CF3, CCl3 or CBr3 to replace the group ##STR6##

The reaction according to step a takes place in the presence of an organic base.

(b) Reaction of the nucleoside-phosphorous acid derivative, of the formula IV, obtained in step a. ##STR7##

The reaction of the compound according to formula IV is carried out with a nucleoside of the general formula V according to claim 1, which is bound to a polymeric carrier. ##STR8## It is possible to use soluble or insoluble, that is to say crosslinked, polymeric carriers, for example modified silica gel, glass, especially "controlled pore glass", polyester, polyamide, polyvinyl alcohol, polysiloxane, polystyrene or the like. Ester bonds are suitable and preferred for the attachment between the carrier and the nucleoside, including those derived from the levulinyl or β-benzoylpropionyl radical; the latter ester bonds can be cleaved with hydrazine under neutral conditions. The acid-labile trityl ether bond, optionally with substituents in the phenyl rings, is also suitable as a method of attachment, compare Liebigs Ann. Chem. 1974, 959.

(c) Oxidation of the carrier-bond nucleotide-nucleoside, of the general formula VI, obtained in step b. ##STR9##

Oxidation leads to a phosphate group; this can be carried out with, for example, iodine/H2 O, H2 O2 or organic peracids or, in general, by oxidation by introduction of O, S or Se.

(d) Blocking of free primary 5'-OH groups which have not been reacted in the reaction according to step b (in the product of the formula V).

These free hydroxyl groups are blocked with a permanent protective group, for example by reaction with acetic anhydride.

(e) Elimination of the protective group(s) R2.

The elimination is carried out using, for example, a protonic acid or Lewis acid, such as ZnBr2 or dialkylaluminum chloride, when R2 represents a trityl group or a methoxy derivative thereof.

(f) Introduction of further nucleoside phosphate or oligonucleoside phosphate units.

Steps a-e can be repeated to introduce at least one nucleoside phosphate moiety. Of course, when oligonucleoside phosphate units are employed, the chains are lengthened by more than one nucleoside phosphate unit.

(g) Elimination of all protective groups.

This elimination can be carried out in such a manner that, using aqueous ammonia, in one step the N-acyl groups on the heterocyclic bases, the ester bond between the oligonucleotide and the carrier (the latter can, where appropriate, also be cleaved with hydrazine under neutral conditions) and the phosphate protective group are eliminated by β-elimination in accordance with the general scheme 1 at the end of the description. An oligonucleotide having only a 5'-terminal trityl protective group is then obtained, and this can be purified directly in a manner known per se, after removal of the volatile base (ammonia), by high-pressure liquid chromatography (HPLC) on reverse phase material.

The intermediates of the general formula IV according to claim 1 are new compounds. They are in the form of very stable compounds which can be prepared in the pure form and are easy to manipulate but nevertheless are very reactive in the sense of forming internucleotide bonds. The use of R3 as a protective group which can be removed by bases via β-elimination makes is possible for the first time to eliminate all the protective groups, apart from the 5'-trityl group, in one step where, in an advantageous manner, by the use of volatile bases the desired oligonucleotide is contaminated with foreign materials to only a very small extent and thus directly afterwards can be purified by reverse phase HPLC due to the hydrophobic 5'-trityl group which is still present.

A further advantage of the process of the invention results from the fact that, due to the removal of the protective group by β-elimination, no attack on the P-atom takes place and thus none of the newly formed internucleotide bonds can be cleaved during the deprotection. Thus, the process of the invention takes very much less time and leads to overall purer products than do the processes hitherto available.

The invention is illustrated in detail below by means of examples, the phosphine derivatives used being those in which R3 is a β-cyanoethyl group. Details of the reaction and physical characteristics of the compounds prepared can be seen in schemes 2 and 3, Table 1, and FIGS. 1-7 at the end of the description.

EXAMPLE 1

Preparation of phosphine derivatives of the general formula III:

β-Cyanoethyl phosphoramidochloridite:

A general summary of the reaction can be seen in scheme 2.

Apart from some improvements, dichloro- -cyanoethoxyphosphine (1) is prepared as in Can. J. Chem. 58, 2686 (1980):

300 ml of ether and 79.0 g (1 mol) of pyridine are added through a dropping funnel to 137.5 g (1.0 mol) of PCl3 in a three-neck flask; the mixture is cooled to -78 C. under argon. Then a solution of 71.0 g (1 mol) of β-cyanoethanol in 150 ml of dry ether is added dropwise over the course of 1 to 1.5 hours. The cooling bath is removed; stirring is continued at room temperature for a further 3 hours (where necessary, another 300 ml of ether are added in order to ensure better stirrability). The stirrer and dropping funnel are removed under argon; the mixture is stored at 0 C. overnight. The solid salts are removed under argon; the precipitate is washed twice with 75 ml of ether each time. The combined organic phases are concentrated in vacuo; the residue is finally distilled in vacuo: boiling point 70-75 C./0.4 mm Hg.

-Cyanoethyl phosphoramidochloridite (3):

A solution of 17.2 g (0.1 mol) of β-cyanoethyl phosphorodichloridite (1) in 60 ml of ether is added dropwise, over the course of 1 to 1.5 hours, to a solution of the N-trimethylsilylated secondary amine (0.1 mol) or secondary amine (0.2 mol) in 30 ml of ether at -20 C. under argon. After stirring at room temperature for 20 hours, the amine hydrochloride is removed; the remaining solution is concentrated. The residue is finally distilled in vacuo in a short-path distillation apparatus.

The physical properties of the compounds thus obtained are summarized in Table 1.

FIGS. 1a, 1b and 1c show 31 P NMR spectra of three different β-cyanoethyl phosphoramidochloridites.

The N-morpholine derivative is too unstable to heat for distillation to be possible. Nevertheless, the preparation is so pure that the residue can be used directly for the preparation of the activated nucleoside derivatives. The purity is usually greater than 95% according to the 31 P NMR spectra.

Nucleoside β-cyanoethyl phosphoramidites:

The preparation of the appropriately protected nucleoside β-cyanoethyl phosphoramidites can be seen in scheme 3.

The synthesis in analogy to Tetrahedron Lett. 22, 1859 (1981), with some improvements, provides good yields.

3.0 mmol of the N-protected 5'-dimethoxytritylated deoxynucleoside are dried azeotropically using THF/toluene, dissolved in 15 ml of dry THF, and 12.0 mmol of N,N,N-diisopropylethylamine are added. 6.0 mmol of the β-cyanoethyl phosphoramidochloridite are added dropwise to the solution under argon, with vigorous stirring, over the course of 2 minutes. After a short time (2 to 5 minutes), the amine hydrochloride precipitates out. The suspension is stirred for a further 30 to 40 minutes. The amine hydrochloride is filtered off under argon and thoroughly washed with dry THF (10 to 15 ml). The entire organic phase is concentrated and dissolved in argon-saturated ethyl acetate (100 ml). The organic phase is extracted twice with 50 ml each time of argon-saturated 10% aqueous sodium carbonate solution. The organic phases are dried with sodium sulfate and evaporated under reduced pressure to give a foam. The foam is dissolved in a little ethyl acetate or toluene and precipitated in n-hexane at -78 C. The activated nucleosides are stable for several months when stored at -20 C. under argon.

FIG. 2 shows the 31 P NMR spectrum of one of the activated deoxynucleosides.

Synthesis of d(CGGTACCG)

100 mg of "controlled pore glass" (CPG) loaded with a total of 8 umol of N-isobutyryldeoxyguanine (compare Tetrahedron Lett. 24, 747 (1983)) are consecutively condensed with the 5'-dimethoxytritylated N-acylated β-cyanoethyl N,N-diisopropylphosphoramidites of the deoxynucleosides C, C, A, T, G, G and C, in each case 20 to 25 equivalents of the phosphoramidite in acetonitrile being activated with 75-80 equivalents of sublimed tetrazole. The condensations are complete within 30 minutes at the most; the coupling yield is greater than 94%. After each condensation, oxidation with I2 /H2 O and blocking of unreacted 5'-OH groups with acetic anhydride are carried out. Then the dimethoxytrityl group is eliminated either with 3% trichloroacetic acid in nitromethane/1% methanol or ZnBr2 /nitromethane/1% H2 O.

The overall yield of the protected octanucleotide at the end of all condensation steps is 55% based on carrier-bound deoxyguanosine.

Complete deprotection and cleavage off from the carrier is achieved in one step by reaction of the glass beads with concentrated aqueous ammonia (3 ml) at 50 C. for 16 hours. The glass beads are then thoroughly washed with 50% aqueous methanol (3 times with 3 ml each time). The liquid phase is removed by evaporation (removal of the methanol) and freeze-drying. Then an aliquot is filtered through a millipore filter and purified by HPLC or RP 18 as can be seen in FIG. 3.

The fractions which contain the 5'-dimethoxytritylated oligonucleotide are collected; the volatile buffer is removed in a rotary evaporator in vacuo. 1 ml of 80% strength acetic acid is added to the residue. After 45 minutes at room temperature, the acetic acid is removed by freeze-drying.

The material thus obtained is phosphorylated in the customary manner (Liebigs Ann. Chem. 1978, 982) with T4-polynucleotide kinase and γ-32 P-ATP. The resulting product is characterized by polyacrylamide gel electrophoresis comparing with a homo-oligo-dT chain length standard (Nucleic Acids Res. 6, 2096 (1979), FIG. 4) and by sequencing according to FIG. 5 (Liebigs Ann. Chem. 1978, 982).

FIGS. 6a to 6c show the results (HPLC, gel electrophoresis, sequencing) of the synthesis of d(GGGATCCC) using the nucleoside β-cyanoethyl N,N-dimethylphosphoramidites. FIGS. 6a to 6c show the results (HPLC, g, electrophoresis, sequencing) of the synthesis of d(GGGATATCCC) using the nucleoside β-cyanoethyl N,N-morpholinophosphoramidites.

The results given in FIGS. 3, 6a and 7a were obtained by using a gradient from 10 to 25 vol. % CH3 CN, 5 min, and 25 to 29 vol. % CH3 CN, 30 min, in 0.1M triethylammonium acetate at pH 7.0.

                                  TABLE 1__________________________________________________________________________Physical data of β-cyanoethyl phosphoramidochloridites    3a             3b             3c.sup.(1)Compound L = N,Ndimethylamino                   L = N,Ndiisopropylamino                                  L = Nmorpholino__________________________________________________________________________Boiling point    90-92/0.6 mm                   103-5/0.08 mm                                  --Chemical shift.sup.(2)    175.97 ppm     179.82 ppm     168.22 ppmin 31 P NMR inCH3 CNChemical shift in    4.01, 4.17 (2t, POCH2, 2H)                   4.02, 4.2 (2t, POCH2, 2H)                                  3.96, 4.1 (2t, POCH2, 2H)1 H NMR in ppm    2.71 (t, CH2CN, 2H)                   3.8 (m, N(CH)2, 2H)                                  3.67 (t, O(CH2)2, 4H)    2.7 (d, N(CH3)2, 6H)                   2.77 (t, CH2 CH, 2H)                                  3.17 (m, PN(CH2)2, 4H)                   1.29 (d, NCH(CH3)2, 12H)                                  2.74 (t, CH2CN 2, 2H) Mass spectrum     ##STR10##                    ##STR11##                                   ##STR12##    (Cl), 136 (C2 H6 N), 110                   (Cl), 166 (C3 H4 NO),                                  (Cl), 152 (C3 H4 NO),    (C3 H4 NO)                   136 (C6 H14 N)                                  136 (C4 H8 O)__________________________________________________________________________ .sup.(1) The crude product after removal of amine hydrochloride and compounds volatile under high vacuum at room temperature has a purity of 93-95% according to the 31 P NMR spectrum .sup.(2) The chemical shifts are determined in acetoned6 with 80% strength H3 PO4 as the external standard. ##STR13##
Patent Citations
Cited PatentFiling datePublication dateApplicantTitle
US4310662 *26 Dec 197912 Jan 1982Genentech, Inc.Nucleosidic phosphorylating agent and methods
US4415732 *27 Mar 198115 Nov 1983University Patents, Inc.Phosphoramidite compounds and processes
US4419509 *24 Aug 19816 Dec 1983Eli Lilly And CompanyProcess for de-cyanoethylating blocked nucleotides
US4458066 *24 Mar 19813 Jul 1984University Patents, Inc.Process for preparing polynucleotides
US4476301 *22 Jun 19829 Oct 1984Centre National De La Recherche ScientifiqueOligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US4500707 *16 Mar 198219 Feb 1985University Patents, Inc.Nucleosides useful in the preparation of polynucleotides
US4591614 *30 Apr 198427 May 1986The Johns Hopkins UniversityPreparation of oligodeoxyribonucleoside alkyl or arylphosphonates
US4605735 *9 Feb 198412 Aug 1986Wakunaga Seiyaku Kabushiki KaishaNon radioactive affinity probe for nucleic acids; immunoassay; genetic engineering
EP0040099A1 *12 May 198118 Nov 1981ens BIO LOGICALS INC.Polynucleotide synthesis
EP0064796A2 *10 May 198217 Nov 1982Unilever N.V.Phosphorylating agent and process for the phosphorylation of organic hydroxyl compounds
EP0090789A1 *24 Mar 19835 Oct 1983Monsanto CompanyChemical DNA synthesis
EP0131993A2 *29 Jun 198423 Jan 1985Stichting Katholieke UniversiteitPhosphorylating agent; a process for the preparation thereof; a process for phosphorylating an organic hydroxyl or amine compound
Non-Patent Citations
Reference
1 *Beaucage and Caruthers (1981) Tet. Lett. 22:1859 1862.
2Beaucage and Caruthers (1981) Tet. Lett. 22:1859-1862.
3 *Caruthers, M. H., Science 230:281 (1985).
4Clesen et al., "Tetrahedron Letters", vol. 25, No. 12, pp. 1307-1310, 1984.
5 *Clesen et al., Tetrahedron Letters , vol. 25, No. 12, pp. 1307 1310, 1984.
6 *Gao, X. et al. Nucleic Acids Research 13(2):573 (1985).
7 *H. Koster et al., Nucleic Acids Research Symposium Series No. 7 (1980) pp. 39 61.
8H. Koster et al., Nucleic Acids Research Symposium Series No. 7 (1980) pp. 39-61.
9Lelsinger et al., "Jour. of the Amer. Chem. Soc." vol. 98, No. 12, Jun. 1976, pp. 3655-3661.
10 *Lelsinger et al., Jour. of the Amer. Chem. Soc. vol. 98, No. 12, Jun. 1976, pp. 3655 3661.
11Marugg et al., "Recl. Trav. Chim. Pays-Bas" vol. 103, pp. 97-98, 1984.
12 *Marugg et al., Recl. Trav. Chim. Pays Bas vol. 103, pp. 97 98, 1984.
13Narang, "Tetrahedron", vol. 39, No. 1. pp. 3-22, 1983.
14 *Narang, Tetrahedron , vol. 39, No. 1. pp. 3 22, 1983.
15 *Ogilvie, K. K. et al., Can J. Chem 58:2686 (1980).
16 *Urdea, M. S. et al. Nucleic Acids Research Symposium Ser. 16 (1985) pp. 257 260.
17Urdea, M. S. et al. Nucleic Acids Research Symposium Ser. 16 (1985) pp. 257-260.
18 *V. Amarnath and A. D. Broom, Chemical Reviews 77(2)183 (1977).
19 *Zon, G. et al. Nucleic Acids Research 13(22):8181 (1985).
Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US4950745 *30 Oct 198721 Aug 1990Daicel Chemical Industries, Ltd.Used in genetic engineering
US4959463 *19 Sep 198625 Sep 1990Genentech, Inc.Coupling ribonucleotides or deoxyribonucleotides though phosphonate groups
US4962193 *28 Dec 19889 Oct 1990Ajinomoto Co., Inc.Adsorption on porous non-polar resin, then separation and elution
US4973679 *18 Sep 198627 Nov 1990University Patents, Inc.Process for oligonucleo tide synthesis using phosphormidite intermediates
US5013830 *12 Feb 19907 May 1991Ajinomoto Co., Inc.Compounds for the cleavage at a specific position of RNA, oligomers employed for the formation of said compounds, and starting materials for the synthesis of said oligomers
US5026838 *4 Aug 198825 Jun 1991Nippon Zeon Co., Ltd.Starting materials for polynucleotides
US5047524 *21 Dec 198810 Sep 1991Applied Biosystems, Inc.Solid phase syntheisis o nonsuelladle porous polystyrene support by phosphoramide or hydrogen phosphonate chemistry
US5134228 *26 Sep 198928 Jul 1992Central Glass Company, LimitedNucleoside-3'-phosphites for synthesis of oligonucleotides
US5151510 *20 Apr 199029 Sep 1992Applied Biosystems, Inc.Method of synethesizing sulfurized oligonucleotide analogs
US5158828 *17 Oct 198927 Oct 1992Sumitomo Metal Industries, Ltd.Metal fibers distributed in carbon matrix; coating or alloying fiber surfaces with metal having equal or less tendency to form carbides; increases bending strength and wear resistance
US5166330 *28 Dec 198724 Nov 1992Hoechst AktiengesellschaftProcess for the preparation of nucleoside alkyl-aralkyl- and aryl-phosphonites and -phosphonates
US5262530 *27 Jul 199016 Nov 1993Applied Biosystems, Inc.Solid phase synthesis on polystyrene support, phosphoramidite chemistry
US5405950 *9 Oct 198711 Apr 1995Amoco CorporationN4 -substituted cytidines and process for incorporation into an oligonucleotides
US5428149 *14 Jun 199327 Jun 1995Washington State University Research FoundationReacting vinyl and aryl stannanes with 2'-deoxy-5-halo-uridine
US5510476 *7 Jul 199423 Apr 1996Isis Pharmaceuticals, Inc.Carbocation scavenging during oligonucleotide synthesis
US5519126 *22 Nov 199321 May 1996University Of Virginia Alumni Patents FoundationCombatting diseases by biochemicl intervention at the rna and dna level
US5571902 *26 May 19945 Nov 1996Isis Pharmaceuticals, Inc.Synthesis of oligonucleotides
US5585481 *14 Jan 199417 Dec 1996Gen-Probe IncorporatedLinking reagents for nucleotide probes
US5591843 *19 Apr 19957 Jan 1997Eaton; Bruce5-modified pyrimidines from palladium catalyzed carbon-carbon coupling
US5594121 *7 Jun 199514 Jan 1997Gilead Sciences, Inc.Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5594122 *19 Sep 199414 Jan 1997Genesys Pharma Inc.Bind to conserved regions of human immunodeficiency virus genetic material to prevent expression thereof
US5597910 *7 Jun 199528 Jan 1997Igen, Inc.Electrochemiluminescent label for DNA probe assays
US5610017 *5 Jun 199511 Mar 1997Igen, Inc.Method for conducting a polymerase chain reaction using an improved electrochemiluminescent label
US5614621 *29 Jul 199325 Mar 1997Isis Pharmaceuticals, Inc.Process for preparing oligonucleotides using silyl-containing diamino phosphorous reagents
US5633361 *21 Mar 199527 May 1997Washington State University Research FoundationOligonucleotides
US5633364 *17 Sep 199227 May 1997Amoco CorporationN4 -substituted 2'-deoxycytidine compounds, oligonucleotides including N4 -labeled 2'-deoxycytidines, and a process for making oligonucleotides with N-modified 2'-deoxycytidines
US5645985 *25 Nov 19928 Jul 1997Gilead Sciences, Inc.Used for diagnosis to detect viruses or diseased conditions
US5646265 *23 Mar 19958 Jul 1997Isis Pharmceuticals, Inc.Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5656744 *7 Jun 199512 Aug 1997Gen-Probe IncorporatedMethods for making nucleotide polymers using novel linking reagents
US5684148 *17 May 19954 Nov 1997Competitive Technologies, Inc.Nucleosides and polynucleotides as fluorescent agents for medical diagnosis
US5696251 *7 Jun 19959 Dec 1997Gen-Probe IncorporatedOligonucleotides
US5705621 *17 Nov 19956 Jan 1998Isis Pharmaceuticals, Inc.Oligomeric phosphite, phosphodiester, Phosphorothioate and phosphorodithioate compounds and intermediates for preparing same
US5714597 *8 Mar 19963 Feb 1998Isis Pharmaceuticals, Inc.Use of carbocation scavenger during oligonucleotide synthesis
US5726300 *15 Jun 199410 Mar 1998Genta IncorporatedForming an internucleoside linkage having a pentavalent phosphorus between a 5'-oxygen of a first nucleoside and a 3'-oxygen of a second nucleoside
US5734041 *20 Oct 199531 Mar 1998Mcgill UniversityPreparation of chiral phosphorothioate oligomers
US5750666 *31 Oct 199412 May 1998Competitve Technologies, Inc.Polynucleotide phosphorodithioate compounds
US5760209 *3 Mar 19972 Jun 1998Isis Pharmaceuticals, Inc.Protecting group for synthesizing oligonucleotide analogs
US5783690 *18 Sep 199721 Jul 1998Isis Pharmaceuticals, Inc.Phosphitylating agents for preparing nucleoside phosphoramidites
US5808035 *4 Dec 199615 Sep 1998Usher; David A.Protected nucleoside and method for its synthesis
US5811537 *14 Jan 199722 Sep 1998Novopharm Biotech Inc.Antisense oligonucleotides targeted against human immunodeficiency virus
US5830653 *7 Jun 19953 Nov 1998Gilead Sciences, Inc.Methods of using oligomers containing modified pyrimidines
US5847106 *27 Jan 19978 Dec 1998Isis Pharmaceuticals Inc.Monomeric and dimeric nucleosides with silyl-containing diamino phosphorous linkages
US5856466 *7 Jul 19975 Jan 1999Isis Pharmaceuticals, Inc.Coupling 2'-o-alkylated guanosine 3'-o-phosphoramidite with a 5'-hydroxyl moiety on an oligonucleotide
US5859232 *31 Oct 199712 Jan 1999Isis Pharmaceuticals, Inc.Process for the synthesis of oligomeric phosphite, phosphodiester, phosphorothioate and phosphorodithioate compounds
US5902881 *3 Mar 199711 May 1999Isis Pharmaceuticals, Inc.Reagent useful for synthesizing sulfurized oligonucleotide analogs
US5914396 *20 Jul 199322 Jun 1999Isis Pharmaceuticals, Inc.2'-O-modified nucleosides and phosphoramidites
US5925732 *2 Jan 199720 Jul 1999Isis Pharmaceuticals, Inc.Chemical reaction apparatus for performing multiple reaction on a surface and collecting the product
US5935527 *29 Apr 199310 Aug 1999The Perkin-Elmer CorporationUsing phosphoramidite or hydrogen phosphonate chemistry on a nonswellable porous polystyrene support
US5945521 *9 Oct 199731 Aug 1999Mcgill UniversityPreparation of phosphorothioate oligomers
US5955591 *13 Mar 199721 Sep 1999Imbach; Jean-LouisPhosphotriester oligonucleotides, amidites and method of preparation
US5959099 *24 Jun 199828 Sep 1999Isis Pharmaceuticals, Inc.Protecting group for synthesizing oligonucleotide analogs
US5959100 *27 Mar 199628 Sep 1999Nexstar Pharmaceuticals, Inc.Pyrimidine nucleosides as therapeutic and diagnostic agents
US6001982 *31 Jul 199614 Dec 1999Isis Pharmaceuticals, Inc.Synthesis of oligonucleotides
US6020475 *10 Feb 19981 Feb 2000Isis Pharmeuticals, Inc.Process for the synthesis of oligomeric compounds
US6031091 *7 Aug 199729 Feb 2000Gen-Probe IncorporatedNon-nucleotide linking reagents for nucleotide probes
US6031092 *15 Oct 199729 Feb 2000Mc Gill UniversityPreparation of phosphorothioate oligomers
US6040438 *25 Jun 199821 Mar 2000Isis Pharmaceuticals, Inc.Contacting oligonucleotide of at least one phosphorous (iii) linkage with solution of solvent and thiodicarbonic acid diester polysulfide in automated dna synthesizer producing oligonucleotide with sulfurized phosphorous (v) linkage
US6043031 *18 Mar 199628 Mar 2000Sequenom, Inc.DNA diagnostics based on mass spectrometry
US6051699 *15 Nov 199618 Apr 2000Isis Pharmaceuticals, Inc.Process for the synthesis of oligomeric compounds
US6060240 *13 Dec 19969 May 2000Arcaris, Inc.Normalizing a gene library by using oligonucleotide tags attached to the cdna inserts, sorting using the tags, and amplifying inserts present at lower abundance in order to match the abundance of all cdna sequences
US6069243 *6 Oct 199830 May 2000Isis Pharmaceuticals, Inc.Process for oligonucleotide synthesis
US6080727 *25 Mar 199727 Jun 2000Istituto Regina ElenaOligonucleotide treatments and compositions for human melanoma
US6111095 *7 Jun 199529 Aug 2000Merck & Co., Inc.Capped synthetic RNA, analogs, and aptamers
US6111251 *19 Sep 199729 Aug 2000Sequenom, Inc.Method and apparatus for MALDI analysis
US6114518 *30 Sep 19995 Sep 2000Becton, Dickinson And CompanyDetecting target nucleic acids; amidation, sulfonamidation, thioamidation
US6121437 *16 Mar 199919 Sep 2000Isis Pharmaceuticals, Inc.Phosphate and thiophosphate protecting groups
US6124445 *9 Jan 199826 Sep 2000Isis Pharmaceuticals, Inc.Phosphotriester oligonucleotides, amidities and method of preparation
US6124450 *27 Jul 199826 Sep 2000Isis Pharmaceuticals, Inc.Processes and intermediates for phosphorous-containing covalent linkages
US6133436 *19 Sep 199717 Oct 2000Sequenom, Inc.Increased surface area for immobilization of nucleic acids; for use in hybridizations
US6133438 *2 Oct 199817 Oct 2000Isis Pharmaceuticals, Inc.Alkylating unblocked cytidine to form 2'-o-alkylated cytidine, blocking the 5'-hydroxyl moiety, and phosphitylating the 3'-position
US6140053 *25 Sep 199831 Oct 2000Sequenom, Inc.Determining a sequence of a nucleic acid
US6143882 *27 Sep 19997 Nov 2000Nexstar Pharmaceuticals, Inc.Reacting a nucleoside starting material containing a leaving group with a vinylstanne of defined formula and carbon monoxide in presence of a palladium catalyst to form an ene-one intermediate, reacting with an amine, and isolating
US6146854 *31 Aug 199514 Nov 2000Sequenom, Inc.Filtration processes, kits and devices for isolating plasmids
US6147200 *19 Aug 199914 Nov 2000Isis Pharmaceuticals, Inc.2'-O-acetamido modified monomers and oligomers
US6153411 *30 Oct 199828 Nov 2000American Water Works Company, Inc.Simple, sensitive, quantitative process for aqueous samples; concentrating organisms present using separation technique, amplifying target nucleic acid using primers of defined nucleotide sequence, detecting amplified nucleic acid acid
US6160109 *30 Jun 199812 Dec 2000Isis Pharmaceuticals, Inc.Preparation of phosphorothioate and boranophosphate oligomers
US6160152 *7 Oct 199912 Dec 2000Isis Pharmaceuticals, Inc.Process for the synthesis of oligomeric compounds
US61691776 Nov 19982 Jan 2001Isis Pharmaceuticals, Inc.Processes for the synthesis of oligomeric compounds
US617221727 Dec 19969 Jan 2001Isis Pharmaceuticals Inc.Method of synthesizing phosphorothioate oligonucleotides
US617500630 Mar 199916 Jan 2001The Perkin-Elmer CorporationAutomated system for polynucleotide synthesis and purification
US619414418 Mar 199627 Feb 2001Sequenom, Inc.Generating two terminated nucleic acids; determination molecular weight; calibration
US61974986 Apr 19996 Mar 2001Sequenom, IncDNA diagnostics based on mass spectrometry
US62073702 Sep 199727 Mar 2001Sequenom, Inc.Determining genotype associated with preferential phenotype; obtain target sequence, prepare target polypeptide, determine mass of the target polypeptide by mass spectrometry and compare to reference, detect allelic variant on chromosome
US620781912 Feb 199927 Mar 2001Isis Pharmaceuticals, Inc.Compounds, processes and intermediates for synthesis of mixed backbone oligomeric compounds
US621135014 Sep 19993 Apr 2001Isis Pharmaceuticals, Inc.Synthesis of oligonucleotides
US62216012 Nov 199924 Apr 2001Sequenom, Inc.DNA diagnostics based on mass spectrometry
US622160515 Feb 200024 Apr 2001Sequenom, Inc.DNA diagnostics based on mass spectrometry
US62216356 May 199924 Apr 2001The Wistar InstituteDetecting preferential nucleic acid in a sample; amplify nucleotide sequences complementary to bound primer; recover amplified products
US622506110 Mar 19991 May 2001Sequenom, Inc.Systems and methods for performing reactions in an unsealed environment
US62254507 Jun 19951 May 2001Sequenom, Inc.Intact positively charged ionized mass-modified nucleic acid molecule
US62354786 Apr 199922 May 2001Sequenom, Inc.DNA diagnostics based on mass spectrometry
US623588714 Nov 199422 May 2001Isis Pharmaceuticals, Inc.Measurement, calibration of oligonucleotide binding to nucleic acids
US62388718 May 200029 May 2001Sequenom, Inc.DNA sequences by mass spectrometry
US623922016 Jul 199929 May 2001Isis Pharmaceuticals, Inc.Recovery of triarylmethyl halide protecting groups cleaved during oligonucleotide synthesis
US62425929 Dec 19995 Jun 2001Isis Pharmaceuticals, Inc.Process for the preparation of 2′-O-alkyl-guanosine, cytidine, and uridine phosphoramidites
US62585386 Apr 199910 Jul 2001Sequenom, Inc.DNA diagnostics based on mass spectrometry
US626225117 Oct 199617 Jul 2001Proligo LlcProduct anchored sequencial synthesis (pass); an anchor group attached to the 5' end of the growing oligonucleotide product allows the coupled product to be separated from unreacted starting materials
US626813115 Dec 199731 Jul 2001Sequenom, Inc.Immobilizing nucleic acid promoter to solid support; hybridization; transcription
US626814415 Sep 199931 Jul 2001Sequenom, Inc.Detecting target nucleotide sequence in sample; obtain nucleotide sequences, immobilize nucleotide sequences on solid support, hybridize probes, ionize and volatize hybridization products, detect sequences by mass spectrometry
US627472523 Oct 199814 Aug 2001Isis Pharmaceuticals, Inc.Providing compound having a hydroxyl group; reacting with phosphitylating reagent in the presence of a pyridinum salt in a solvent under conditions of time, temperature and pressure to yield phosphitylated compound
US627733418 May 199921 Aug 2001Isis Pharmaceuticals, Inc.Chemical synthesis apparatus employing a droplet generator
US62775736 Apr 199921 Aug 2001Sequenom, Inc.Detecting nucleic acid molecules and sequences for diagnosis of genetic disease or chromosomal abnormality, a predisposition to a disease, or infection by a pathogenic organism; high speed and accurate
US62911705 Apr 199918 Sep 2001Board Of Trustees Of Leland Stanford UniversityComplementary deoxyribonucleic acid is synthesized by rna using a complementary primer linked to a rna polymerase promoter, anti-sense rna is transcribed from the cdna when rna polymerase binds the promoter region; diagnosis/forensic
US629466426 May 199525 Sep 2001Isis Pharmaceuticals, Inc.Preparing oligonucleotide blocks having common subsequences, phospitylating, and performing solid phase synthesis (sps) to form target oligonucleotides wherein the synthons used in sps comprise phosphitylated oligonucleotide blocks
US630007631 Jan 20009 Oct 2001Sequenom, Inc.Diagnostic detecting method of target nucleic acid in biological sample by amplifying nucleic acid, ionizing and volatilizing the product then analyze nucleic acid by mass spectrometry to detect presence of nucleic acid in the sample
US63066473 Dec 199323 Oct 2001Ajinomoto Co., Inc.Process for producing and purifying 2′,3′-dideoxynucleosides, and process for producing 2′,3′-dideoxy-2′,3′-didehydronucleosides
US63229702 Sep 199827 Nov 2001Sequenom, Inc.Mass spectrometric detection of polypeptides
US63264788 Jul 19984 Dec 2001Isis Pharmaceuticals, Inc.Oligonucleotides and medical diagnosis, metabolism
US63264794 Jan 19994 Dec 2001Boston Probes, Inc.Synthetic polymers and methods, kits or compositions for modulating the solubility of same
US633543424 Mar 19991 Jan 2002Isis Pharmaceuticals, Inc.,Nucleosidic and non-nucleosidic folate conjugates
US63354377 Sep 19981 Jan 2002Isis Pharmaceuticals, Inc.Methods for the preparation of conjugated oligomers
US633543911 Jun 19981 Jan 2002Isis Pharmaceuticals, Inc.Method of preparing phosphoramidites
US638036812 Feb 199630 Apr 2002Isis Pharmaceuticals, Inc.Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US638037823 Dec 199930 Apr 2002Toagosei Company, Ltd.Biosynthesis
US638762818 Sep 200014 May 2002Sequenom, Inc.Mass spectrometric detection of polypeptides
US639551718 Jul 200028 May 2002American Water Works Company, Inc.Methods and kits for detection of cryptosporidium parvum
US63997568 Jul 19994 Jun 2002Isis Pharmaceuticals, Inc.Process for the synthesis of oligomeric compounds
US639976517 Mar 19994 Jun 2002Isis Pharmaceuticals, Inc.Dealkylation using acid solution
US63998311 Dec 19994 Jun 2002Isis Pharmaceuticals, Inc.Thiodicarbonic acid diester polysulfide
US64037818 Jan 200111 Jun 2002Isis Pharmaceuticals, Inc.Method of synthesizing phosphorothioate oligonucleotides
US64141357 Jul 19992 Jul 2002Isis Pharmaceuticals, Inc.Phosphonylation of alkylnucleoside with bis(trimethylsilyl)phosphonite in base presence; concentration; adding methanol solvent; nuclease resistance; hybridization
US64205516 Mar 199816 Jul 2002Viventia Biotech Inc.Antisense oligonucleotides targeted against human immunodeficiency virus
US642396614 Jan 200023 Jul 2002Sequenom, Inc.Method and apparatus for maldi analysis
US64289556 Nov 19966 Aug 2002Sequenom, Inc.DNA diagnostics based on mass spectrometry
US644094314 Jul 199927 Aug 2002Isis Pharmaceuticals, Inc.Mimic and/or modulate the activity of wild-type nucleic acids; the 5' and the 3'-terminal internucleoside linkages are chirally sp and internal internucleoside linkages are chirally rp; resistant to nuclease degradation; psoriasis
US64411616 Nov 200027 Aug 2002Gilead Sciences, Inc.Urea nucleosides as therapeutic and diagnostic agents
US646218429 Nov 20008 Oct 2002Isis Pharmaceuticals, Inc.Compounds, processes and intermediates for synthesis of mixed backbone oligomeric compounds
US64656289 Apr 199915 Oct 2002Isis Pharmaceuticals, Inc.Process for the synthesis of oligomeric compounds
US647621618 Oct 19965 Nov 2002Mcgill UniversityPreparation of phosphorothioate oligomers
US64859132 Oct 200026 Nov 2002Sequenom, Inc.Systems and methods for performing reactions in an unsealed environment
US64863122 Apr 200126 Nov 2002Isis Pharmaceuticals, Inc.Synthesis of oligonucleotides
US650062128 Feb 200131 Dec 2002Sequenom, Inc.Detection uisng mass spectrometry
US652177511 Dec 200118 Feb 2003Isis Pharmaceuticals, Inc.Genetically engineered oligonucleotides for use as diagnostic and therapeutic tools in gene therapy
US652863116 Jun 19984 Mar 2003Isis Pharmaceuticals, Inc.Folates are conjugated to an oligonucleotide including at the 2'-, 3'-, 5'-, nucleobase and internucleotide linkage sites; improved transfer across cellular membranes; regioselective synthesis
US653159024 Apr 199811 Mar 2003Isis Pharmaceuticals, Inc.Processes for the synthesis of oligonucleotide compounds
US65346397 Jul 200018 Mar 2003Isis Pharmaceuticals, Inc.Guanidinium functionalized oligonucleotides and method/synthesis
US655863321 Sep 19946 May 2003Isis Pharmaceuticals, Inc.Utilizing jetting droplet generation and direction techniques; easy, economical, efficient, high yield
US65589027 May 19996 May 2003Sequenom, Inc.Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of macromolecules
US656938528 Oct 199927 May 2003Sequenom, Inc.Fluid dispensing apparatus for a multiwell substrate; Serial and parallel dispensing tools that can deliver defined and controlled volumes of fluid to generate multi-element arrays of sample material on a substrate surface
US658948511 Jun 20018 Jul 2003Sequenom, Inc.Solid support for use in the diagnosis of preferential genetic disorders
US65934667 Jul 199915 Jul 2003Isis Pharmaceuticals, Inc.Guanidinium functionalized nucleotides and precursors thereof
US65968571 Dec 199922 Jul 2003Mcgill UniversityPreparation of phosphorothioate oligomers
US66000326 Aug 199929 Jul 2003Isis Pharmaceuticals, Inc.Compound for use in the treatment of viral, protozoan and parasitic infections
US660266228 Nov 20005 Aug 2003Sequenom, Inc.Determining whether a target nucleotide is present in a nucleic acid molecule; contacting hybridized primer with deoxyribonucleoside triphosphates, chain terminating nucleotides and a DNA polymerase; diagnosing genetic diseases
US661083716 Mar 200026 Aug 2003Isis Pharmaceuticals, Inc.Preparation of an oligonucleotide compound of given formula by reacting with given compounds, followed by oxidation or sulfurization
US66108426 May 199926 Aug 2003Isis Pharmaceuticals, Inc.Removing hydroxyl protecting group from compound of given formula and treating with another compound; wide range of diagnostic and therapeutic uses
US66390617 Jul 199928 Oct 2003Isis Pharmaceuticals, Inc.C3′-methylene hydrogen phosphonate oligomers and related compounds
US664237312 Dec 20024 Nov 2003Isis Pharmaceuticals, Inc.Activators for oligonucleotide synthesis
US664611411 Oct 200211 Nov 2003Isis Pharmaceuticals, Inc.Especially phosphorothioate oligonucleotides, and to intermediates used in that process; solution phase synthesis having improved efficiencies and enhanced convenience and cost
US667391211 Apr 20026 Jan 2004Isis Pharmaceuticals, Inc.2′-O-aminoethyloxyethyl-modified oligonucleotides
US66774718 Nov 200213 Jan 2004Isis Pharmaceuticals, Inc.Intermediates for the synthesis of oligonucleotide analogues
US6693187 *17 Oct 200017 Feb 2004Lievre Cornu LlcEnhancing nuclease resistance
US670653010 May 199916 Mar 2004Sequenom, Inc.IR-MALDI mass spectrometry of nucleic acids using liquid matrices
US67235647 May 199820 Apr 2004Sequenom, Inc.IR MALDI mass spectrometry of nucleic acids using liquid matrices
US676229823 Feb 200113 Jul 2004The United States Of America As Represented By The Department Of Health And Human ServicesThermolabile phosphorus protecting groups, associated intermediates and methods of use
US677044230 Nov 20013 Aug 2004Boston Probes, Inc.Methods for modulating the solubility of synthetic polymers
US678098917 Jan 200324 Aug 2004Isis Pharmaceuticals, Inc.Hybrid oligonucleotide
US679414125 Oct 200121 Sep 2004Arcturus Bioscience, Inc.Nucleic acid amplification
US679450222 Mar 200221 Sep 2004Isis Pharmaceuticals, Inc.Half-life is determined for deprotection of 5'-oh protecting group at the 5'-terminus of an oligonucleotide post-synthesis
US68124553 Apr 20022 Nov 2004Sequenom, Inc.Method and apparatus for MALDI analysis
US68183946 Nov 199716 Nov 2004Sequenom, Inc.High density immobilization of nucleic acids
US68183956 Nov 200016 Nov 2004California Institute Of TechnologyFor automatic high speed/throughput analysis without size-separating dna fragments on polyacrylamide gel electrophoresis
US682208816 Jul 200223 Nov 2004Isis Pharmaceuticals, Inc.Synthesis of oligonucleotides on solid support
US682533830 Mar 200130 Nov 2004Isis Pharmaceuticals, Inc.Selective functionalization of oligonucleotides with conjugate groups without the need for post-synthetic labeling; solid phase synthesis; deblocking an acid labile hydroxyl group and reacting with a phosphoramidite
US68469228 Nov 200025 Jan 2005Isis Pharmaceuticals, Inc.Activators for oligonucleotide synthesis
US68615149 Oct 20011 Mar 2005Isis Pharmaceuticals, Inc.The folate can be attached via the alpha - or gamma -carboxylate, optionally through a linking group.
US686151824 Sep 20021 Mar 2005Mcgill UniversityHybrid oligonucleotide
US686729412 Nov 199915 Mar 2005Isis Pharmaceuticals, Inc.Design and synthesis of nuclease resistant gapped oligonucleotides which are useful for therapeutics, diagnostics and as research reagents ; are also capable of modulating the activity of DNA and RNA
US687003919 Sep 200322 Mar 2005Isis Pharmaceuticals, Inc.Synthesizing phosphorothioate oligonucleotides and their intermediates; used in genomic research as probes, primers and alsoin diagnositics
US687559314 Nov 20025 Apr 2005Isis Pharmaceuticals, Inc.Oligomers capable of forming triplexes with various target sequences such as virus or oncogene sequences by coupling into major groove of target DNA duplex or forming duplexes by binding to single-stranded DNA or RNA encoded by target genes
US689415824 Feb 200317 May 2005Honeywell International Inc.Reacting nucleoside with cyanoethyl-N,N,N',N'-tetraisopropyl-phosphorodiamidite in presence of trifluoroacetic acid-diisopropylethylamine complex
US690618230 Nov 200114 Jun 2005Cell Works Therapeutics, Inc.Inhibiting abnormal cellular proliferation in a mammal; and a method of inhibiting replication of a virus in a mammal.
US691134518 Jul 200128 Jun 2005California Institute Of TechnologyMethods and apparatus for analyzing polynucleotide sequences
US69141389 Aug 20025 Jul 2005Gilead Sciences, Inc.Urea nucleosides as therapeutic and diagnostic agents
US691414820 Sep 20025 Jul 2005Isis Pharmaceuticals, Inc.Monomer for use in huaman therapeutics and diagnostics
US691943710 Jun 199919 Jul 2005Isis Pharmaceuticals, Inc.A prodrug forms of oligonucleotides, able releasing the oligonucleotide by intercellular enzymes, a protecting groups comprising a nucleoside ribose esters, or binding to polyamides, or polyethers, or a solid supports
US694963325 Aug 199827 Sep 2005Sequenom, Inc.Primers useful for sizing nucleic acids
US696278318 Dec 20018 Nov 2005Isis Pharmaceuticals, Inc.Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US696504116 Feb 200015 Nov 2005The United States Of America As Represented By The Department Of Health And Human ServicesN-acylphosphoramidites and their use in oligonucleotide synthesis
US696724724 Jul 200222 Nov 2005Isis Pharmaceuticals, Inc.Deprotection of phosphorus in oligonucleotide synthesis
US697486518 Dec 200213 Dec 2005Isis Pharmaceuticals, Inc.C3′ -methylene hydrogen phosphonate oligomers and related compounds
US699190330 Apr 200231 Jan 2006Sequenom, Inc.Solid phase sequencing of double-stranded nucleic acids
US700200612 Feb 200321 Feb 2006Isis Pharmaceuticals, Inc.reacting a nucleoside with a protecting reagent and activator to produce a regioselectively protected oligonucleoside; lutidine activator improves selectivity; for automatic synthesis of primers, probes and antiseisure agents
US704181620 Jan 20049 May 2006Isis Pharmaceuticals, Inc.solid phase deprotection of cyanoethyl groups on the phosphorothioate backbone of an oligonucleotide with an amine deprotecting agent (triethylamine); cleaving the oligonucleotide from the support; minimal acrylonitrile nucleobase adduct formation; by-product inhibition
US704910215 Nov 200023 May 2006Board Of Trustees Of Leland Stanford UniversityMulti-gene expression profile
US70570278 Dec 20036 Jun 2006Isis Pharmaceuticals, Inc.primers/probes for amplification and detection of nucleic acids; for treatment of viral diseases
US7067641 *24 Nov 200327 Jun 2006Lievre Cornu LlcPhosphinoamidite carboxylates and analogs thereof in the synthesis of oligonucleotides having reduced internucleotide charge
US707456324 Feb 200311 Jul 2006Sequenom, Inc.Mass spectrometric methods for detecting mutations in a target nucleic acid
US709832622 Jan 200329 Aug 2006Sigma-Aldrich Co.Methods for the integrated synthesis and purification of oligonucleotides
US71189075 Jun 200210 Oct 2006Li-Cor, Inc.Multichannel microfluidic system for sequencing preferential nucleotide sequences
US712594519 Sep 200324 Oct 2006Varian, Inc.Chromatographic separation of a mixture of analytes by using an absorber, a halogenated polymers of vinyl aromatic monomers comprising styrene and divinylbenzene; separating polar analytes from nonpolar analytes; separating labeled nucleic acids from unlabeled nucleic acids
US713556527 Jul 200114 Nov 2006Agilent Technologies, Inc.Synthesis of polynucleotides using combined oxidation/deprotection chemistry
US716956024 May 200430 Jan 2007Helicos Biosciences CorporationShort cycle methods for sequencing polynucleotides
US716991631 Mar 200330 Jan 2007Isis Pharmaceuticals, Inc.Chloral-free DCA in oligonucleotide synthesis
US718682228 Dec 20046 Mar 2007Isis Pharmaceuticals, Inc.Process for the synthesis of oligomeric compounds
US719889310 Oct 20003 Apr 2007Sequenom, Inc.DNA diagnostics based on mass spectrometry
US719923614 Sep 20043 Apr 2007Isis Pharmaceuticals, Inc.Deprotecting nucleotides having phosphotriester or phosphorodithioate linkages and at least one cyanoethyl protecting group by reaction with an amine deprotecting agent; solid phase synthesis
US721781416 Nov 200415 May 2007Honeywell International Inc.Reacting nucleoside with cyanoethyl-N,N,N',N'-tetraisopropyl-phosphorodiamidite in presence of trifluoroacetic acid-diisopropylethylamine complex
US722054930 Dec 200422 May 2007Helicos Biosciences CorporationStabilizing a nucleic acid for nucleic acid sequencing
US722701628 Dec 20045 Jun 2007Isis Pharmaceuticals, Inc.Process for the synthesis of oligomeric compounds
US725587421 Dec 200114 Aug 2007Closure Medical CorporationUse as sealants and adhesives for medical uses, and as polymeric formulations for biocompatible implants and matrices and drug delivery
US726217713 Jun 200528 Aug 2007Cell Works Therapeutics, Inc.Conjugates of glycosylated/galactosylated peptide, bifunctional linker, and nucleotidic monomers/polymers, and related compositions and methods of use
US727393326 Feb 199825 Sep 2007Isis Pharmaceuticals, Inc.Methods for synthesis of oligonucleotides
US72765992 Jun 20042 Oct 2007Isis Pharmaceuticals, Inc.Oligonucleotide synthesis with alternative solvents
US728542223 Jan 199723 Oct 2007Sequenom, Inc.Systems and methods for preparing and analyzing low volume analyte array elements
US729146030 May 20036 Nov 2007Verenium CorporationIdentifying and characterizing restriction fragment length polymorphisms associated with sensitivity to advevrse drug reactions
US729751812 Mar 200220 Nov 2007California Institute Of TechnologyMethods and apparatus for analyzing polynucleotide sequences by asynchronous base extension
US731900324 Feb 199815 Jan 2008The Trustees Of Boston UniversityArrays of probes for positional sequencing by hybridization
US73550373 Dec 20028 Apr 2008The United States Of America As Represented By The Department Of Health And Human ServicesThermolabile hydroxyl protecting groups and methods of use
US73975468 Mar 20068 Jul 2008Helicos Biosciences CorporationSystems and methods for reducing detected intensity non-uniformity in a laser beam
US739984527 Jan 200715 Jul 2008Isis Pharmaceuticals, Inc.For modulating gene expression pathways, including those relying on mechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as well as other antisense mechanisms based on target degradation or target occupancy; of therapeutic, diagnostics, and research applications; nuclease resistance
US741978711 May 20062 Sep 2008Sequenom, Inc.Amplifying, ionizing and volatilizing for determining presence of nucleotide sequence; matrix-assisted laser desorption, ionization time-of-flight; genetic disorders
US742767522 Aug 200523 Sep 2008Isis Pharmaceuticals, Inc.Compounds and methods for the characterization of oligonucleotides
US74276781 May 200123 Sep 2008Sigma-Aldrich Co.Reacting a derivatized molecule with a support; diels-alder reaction; fluorescein; diene or dienophile
US746244914 May 20049 Dec 2008California Institute Of TechnologyUsing immobilized microfluidic chip as tool in sequencing of preferential nucleotide sequences; high throughput assay
US74767346 Dec 200513 Jan 2009Helicos Biosciences CorporationNucleotide analogs
US748212028 Jan 200527 Jan 2009Helicos Biosciences CorporationMethods and compositions for improving fidelity in a nucleic acid synthesis reaction
US749149826 Oct 200617 Feb 2009Helicos Biosciences CorporationShort cycle methods for sequencing polynucleotides
US75012512 Oct 200610 Mar 2009Sequenom, Inc.DNA diagnostics based on mass spectrometry
US754768410 May 200716 Jun 2009Isis Pharmaceuticals, Inc.(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphinoxy]-1-[1-(S)-(4,4'-dimethoxytrityl)oxy-ethyl]-3-(uracil-1-yl)-2,5-dioxa-bicyclo[2.2.1]heptane; useful for enhancing properties of oligomeric compounds including for example enhanced nuclease resistance
US756968610 May 20074 Aug 2009Isis Pharmaceuticals, Inc.Compounds and methods for synthesis of bicyclic nucleic acid analogs
US757945912 Dec 200525 Aug 2009Ngo Nam QActivator bound solid supports for nucleic acid synthesis via the phosphoramidite approach
US76084339 Feb 200527 Oct 2009Idexx LaboratoriesMultiplex real-time RT-PCR assay with a heterologous internal control system; could be performed as a one-tube assay without loss of sensitivity or specificity
US761562931 Dec 200210 Nov 2009Sigma-Aldrich Co.Methods and compositions for the tandem synthesis of two or more oligonucleotides on the same solid support
US763556225 May 200522 Dec 2009Helicos Biosciences CorporationLabelling, hybridization, solid phase synthesis; attaching nucleic acid molecules via covalent linkages to reactive chemical groups on surface, blocking with solution comprising potassium phosphate, reacting with label, rinsing, then analyzing
US76455965 May 200412 Jan 2010Arizona Board Of RegentsPolymerase chain reactions; exonucleases; microfluidics
US765907026 Oct 20079 Feb 2010Pacific Biosciences Of California, Inc.Charge switch nucleotides
US766659326 Aug 200523 Feb 2010Helicos Biosciences Corporationcapturing with probe, annealing primer, adding polymerase, removing unincorporated nucleotides; sonication; point mutations
US76668549 Dec 200823 Feb 2010Isis Pharmaceuticals, Inc.Bis-modified bicyclic nucleic acid analogs
US769131612 Feb 20046 Apr 2010Chemistry & Technology For Genes, Inc.Devices and methods for the synthesis of nucleic acids
US769590220 Feb 200213 Apr 2010Isis Pharmaceuticals, Inc.Contacting test solution containing Rna and enzyme; hybridization
US774145729 May 200822 Jun 2010Isis Pharmaceuticals, Inc.6-modified bicyclic nucleic acid analogs
US77451168 Apr 200429 Jun 2010Pacific Biosciences Of California, Inc.Polymerase-nucleic acid complex comprising anchor which irreversibly associates target nucleotide with multimeric complex for use as tool in enhancing polymerase processivity in DNA sequencing
US775013119 Mar 20096 Jul 2010Isis Pharmaceuticals, Inc.(1R,3R,4R,7S)-7-[2-Cyanoethoxy(diisopropylamino)phosphinoxy]-1-[1-(S)-(4,4'-dimethoxytrityl)oxy-ethyl]-3-(uracil-1-yl)-2,5-dioxa-bicyclo[2.2.1]heptane; useful for enhancing properties of oligomeric compounds including nuclease resistance
US775906522 May 200820 Jul 2010Sequenom, Inc.Digesting a target nucleic acid molecule; capturing digested fragments on a solid support that containing oligonucleotides complementary, detecting hybrids and molecular weight of captured fragments by mass spectrometry to identify mutations; Fast and highly accurate
US775948029 Jan 200720 Jul 2010Isis Pharmaceuticals, Inc.Chloral-free DCA in oligonucleotide synthesis
US777243925 Oct 200510 Aug 2010Operon Biotechnologies, Inc.compounds comprising a phosphoramidite moiety and a protecting group bonded to a linker chain, and reporter group bonded to the protecting group, used for the functionalization, derivatization and modification that can be introduced into nucleotides
US780352914 Sep 199928 Sep 2010Sequenom, Inc.Hybridizing the nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes with a single-stranded portion having a variable region; molecular weight determined by mass spectrometry; target sequence determined from fragment molecular weights
US78121494 Nov 200312 Oct 2010Isis Pharmaceuticals, Inc.Comprises nucleotide sequences for use in gene expression inhibition, viral defense, transposon silencing and treatment of metabolic disorders; antisense agents
US785831123 Oct 200728 Dec 2010Pacific Biosciences Of California, Inc.Polymerase-nucleic acid complex comprising anchor which irreversibly associates target nucleotide with multimeric complex for use as tool in enhancing polymerase processivity in DNA sequencing; detecting incorporation of a labeled nucleotide triphosphate onto the growing end of a primer nucleic acid
US785856016 Jul 200228 Dec 2010Caprotec Bioanalytics GmbhCapture compounds, collections thereof and methods for analyzing the proteome and complex compositions
US787573320 Sep 200425 Jan 2011Isis Pharmaceuticals, Inc.Oligomeric compounds comprising 4′-thionucleosides for use in gene modulation
US78840867 Sep 20058 Feb 2011Isis Pharmaceuticals, Inc.identifying siRNA and double-stranded RNA compounds, having bioactivity in vivo; nucleosides
US789734513 Feb 20091 Mar 2011Helicos Biosciences CorporationShort cycle methods for sequencing polynucleotides
US793925623 Oct 200710 May 2011Pacific Biosciences Of California, Inc.Composition and method for nucleic acid sequencing
US793967731 Aug 201010 May 2011Isis Pharmaceuticals, Inc.Oligomeric compounds comprising 4′-thionucleosides for use in gene modulation
US796052630 Mar 200914 Jun 2011Berry And Associates, Inc.Colorimetric-oxycarbonyl protecting groups for use in organic syntheses
US79816049 Feb 200519 Jul 2011California Institute Of TechnologyMethods and kits for analyzing polynucleotide sequences
US80221935 May 201020 Sep 2011Isis Pharmaceuticals, Inc.6-modified bicyclic nucleic acid analogs
US803046731 Mar 20104 Oct 2011Isis Pharmaceuticals, Inc.5′-modified bicyclic nucleic acid analogs
US808874614 Jan 20103 Jan 2012Isis Pharmaceuticals, Inc.Bis-modified bicyclic nucleic acid analogs
US808890415 Aug 20083 Jan 2012Isis Pharmaceuticals, Inc.Tetrahydropyran nucleic acid analogs
US814851610 Aug 20103 Apr 2012Pacific Biosciences Of California, Inc.Flowcell systems for single molecule detection
US815360220 May 199910 Apr 2012Isis Pharmaceuticals, Inc.Composition and methods for the pulmonary delivery of nucleic acids
US81880597 May 200729 May 2012Isis Pharmaceuticals, Inc.Compounds and methods for modulating expression of GCGR
US826898023 Sep 201118 Sep 2012Isis Pharmaceuticals, Inc.5′-modified bicyclic nucleic acid analogs
US82782831 Jul 20082 Oct 2012Isis Pharmaceuticals, Inc.6-disubstituted or unsaturated bicyclic nucleic acid analogs
US827842522 May 20082 Oct 2012Isis Pharmaceuticals, Inc.N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
US82784266 Jun 20082 Oct 2012Isis Pharmaceuticals, Inc.Carbocyclic bicyclic nucleic acid analogs
US836223215 Sep 201029 Jan 2013Isis Pharmaceuticals, Inc.Compounds and methods for modulating expression of SGLT2
US836223426 Mar 200729 Jan 2013Archemix LlcSolid support reagents for synthesis
US83729677 May 200712 Feb 2013Isis Pharmaceuticals, Inc.Compounds and methods for modulating expression of GCCR
US83949473 Mar 200412 Mar 2013Isis Pharmaceuticals, Inc.has a plurality of modified ribofuranosyl nucleosides at defined locations; comprise double stranded constructs wherein one of the strands capable of hybridizing to a nucleic acid target
US840483015 Dec 201026 Mar 2013Somalogic, Inc.Method for generating aptamers with improved off-rates
US842637819 Mar 200923 Apr 2013Isis Pharmaceuticals, Inc.Oligomeric compounds comprising tricyclic nucelosides and methods for their use
US84408031 Dec 201114 May 2013Isis Pharmaceuticals, Inc.Tetrahydropyran nucleic acid analogs
US847096621 Jun 201025 Jun 2013Protelica, Inc.Universal fibronectin type III binding-domain libraries
US850180523 Sep 20096 Aug 2013Isis Pharmaceuticals, Inc.Substituted alpha-L-bicyclic nucleosides
US85306406 Feb 200910 Sep 2013Isis Pharmaceuticals, Inc.Bicyclic cyclohexitol nucleic acid analogs
US85363201 Feb 201017 Sep 2013Isis Pharmaceuticals, Inc.Tetrahydropyran nucleic acid analogs
US854655621 Nov 20081 Oct 2013Isis Pharmaceuticals, IncCarbocyclic alpha-L-bicyclic nucleic acid analogs
US85694744 Mar 200529 Oct 2013Isis Pharmaceuticals, Inc.Double stranded constructs comprising one or more short strands hybridized to a longer strand
US86041834 Nov 200310 Dec 2013Isis Pharmaceuticals, Inc.Compositions comprising alternating 2′-modified nucleosides for use in gene modulation
US860419223 Sep 200910 Dec 2013Isis Pharmaceuticals, Inc.Cyclohexenyl nucleic acids analogs
US863697815 Jun 200928 Jan 2014The United States Of America, As Represented By The Secretary, Department Of Health And Human ServicesCyclized NGR peptide compounds, compositions, and methods of their use
US86738717 May 200718 Mar 2014Isis Pharmaceuticals, Inc.Compounds and methods for modulating expression ApoB
US868001918 Sep 200925 Mar 2014Protelica, Inc.Universal fibronectin Type III binding-domain libraries
US86976084 Aug 201015 Apr 2014Protelica, Inc.Universal fibronectin type III binding-domain libraries
US87037288 Sep 200422 Apr 2014Isis Pharmaceuticals, Inc.Gapped oligomeric compounds having linked bicyclic sugar moieties at the termini
US8715927 *22 Apr 20096 May 2014Oregon Health & Science UniversityInhibition of DNA polymerases to augment chemotherapeutic and antimicrobial agents
US87164672 Mar 20116 May 2014Gen9, Inc.Methods and devices for nucleic acid synthesis
US87589956 Aug 201024 Jun 2014Sequenom, Inc.Solid phase sequencing of biopolymers
US87791186 Jan 201115 Jul 2014Isis Pharmaceuticals, Inc.Base modified bicyclic nucleosides and oligomeric compounds prepared therefrom
US20110236507 *22 Apr 200929 Sep 2011Oregon Health & Science UniversityInhibition of dna polymerases to augment chemotherapeutic and antimicrobial agents
USRE35609 *9 Feb 199516 Sep 1997Ajinomoto Co., Inc.Process for purifying 2',3'-dideoxynucleosides
USRE39464 *24 Aug 20049 Jan 2007Isis Pharmaceuticals Inc.Oligonucleolotides having site specific chiral phosphorothioate internucleoside linkages
USRE4100517 Oct 200224 Nov 2009Sequenom, Inc.Functionalized beads for the immobilization of nucleic acids, wherein the beads are stably associated with a solid support selected from multiwell plates, arrays of pits and multiwell supports
USRE4309718 May 201010 Jan 2012Illumina, Inc.Massively parallel signature sequencing by ligation of encoded adaptors
USRE4469322 Jul 20097 Jan 2014Sequenom, Inc.Beads bound to a solid support and to nucleic acids
EP1476407A2 *24 Feb 200317 Nov 2004Honeywell International Inc.Methods of producing phosphitylated compounds
EP1657306A116 Nov 200417 May 2006Qiagen GmbHGene silencing using sense DNA and antisense RNA hybrid constructs coupled to peptides facilitating the uptake into cells
EP1724348A212 Oct 199522 Nov 2006Solexa, Inc.Molecular tagging system
EP1967592A16 Jun 199610 Sep 2008Solexa, Inc.Method of improving the efficiency of polynucleotide sequencing
EP2053406A216 Jul 200229 Apr 2009caprotec bioanalytics GmbHCapture compounds, collections thereof and methods for analyzing the proteome and complex compositions
EP2226330A17 Jun 20018 Sep 2010Pacific Biosciences of California, Inc.Charge-switch nucleotides
EP2298786A116 Apr 199623 Mar 2011Solexa, Inc.DNA sequencing by parallel oligonucleotide extensions
EP2298787A116 Apr 199623 Mar 2011Solexa, Inc.DNA sequencing by parallel oligonucleotide extensions
EP2314594A127 Jan 200727 Apr 2011Isis Pharmaceuticals, Inc.6-modified bicyclic nucleic acid analogs
EP2316974A18 May 20014 May 2011Biosearch Technologies, Inc.Dark quenchers for donor-acceptor energy transfer
EP2332951A227 Jan 200715 Jun 2011ISIS Pharmaceuticals, Inc.6-modified bicyclic nucleic acid analogs
EP2333113A18 May 200115 Jun 2011Biosearch Technologies, Inc.Dark quenchers for donor-acceptor energy transfer
EP2634263A17 Jun 20014 Sep 2013Pacific Biosciences of California, Inc.Charge-switch nucleotides based method for sequencing of nucleic acids
EP2708541A115 Nov 200419 Mar 2014Isis Pharmaceuticals, Inc.5,6-dihydroxy-isoindole derivatives as linkers for oligomer solid phase synthesis
WO1989009221A1 *22 Mar 19895 Oct 1989Univ VirginiaOligonucleotide n-alkylphosphoramidates
WO1989009780A1 *12 Apr 198919 Oct 1989Chemical Industry Inst Of ToxiSite-specific tritium-labeled oligodeoxynucleotides
WO1991016331A1 *14 Feb 199131 Oct 1991Applied BiosystemsMethod of synthesizing sulfurized oligonucleotide analogs
WO1992007864A1 *21 Oct 199127 Apr 1992Genta IncImproved process for the synthesis of oligomers
WO1993012135A111 Dec 199224 Jun 1993Gilead Sciences IncNuclease stable and binding competent oligomers and methods for their use
WO2000002896A1 *8 Jul 199920 Jan 2000Zacharia S CheruvallathImproved process for the synthesis of oligonucleotides and analogues thereof
WO2002027036A224 Sep 20014 Apr 2002Boston Probes IncProbes, probe sets, methods and kits pertaining to the detection, identification and/or enumeration of bacteria
WO2003072586A2 *24 Feb 20034 Sep 2003Honeywell Int IncMethods of producing phosphitylated compounds
WO2003088911A216 Apr 200330 Oct 2003Roberto CreaUniversal libraries for immunoglobulins
WO2004009612A1 *17 Jul 200329 Jan 2004Isis Pharmaceuticals IncDeprotection of phosphorus in oligonucleotide synthesis
WO2006047628A2 *25 Oct 20054 May 2006Operon Holdings IncAmino or thiol linker building block for the synthesis of amino- or thiol-functionalized nucleic acids and methods of making and use thereof
WO2007134181A210 May 200722 Nov 2007Isis Pharmaceuticals Inc5'-modified bicyclic nucleic acid analogs
WO2008150729A222 May 200811 Dec 2008Isis Pharmaceuticals IncN-substituted-aminomethylene bridged bicyclic nucleic acid analogs
WO2010064146A22 Dec 200910 Jun 2010Chiralgen, Ltd.Method for the synthesis of phosphorus atom modified nucleic acids
WO2010090857A221 Jan 201012 Aug 2010Vertex Pharmaceuticals IncorporatedMethods for amplifying hepatitis c virus nucleic acids
WO2010090969A11 Feb 201012 Aug 2010Isis Pharmaceuticals, Inc.Tetrahydropyran nucleic acid analogs
WO2011005860A27 Jul 201013 Jan 2011Alnylam Pharmaceuticals, Inc.5' phosphate mimics
WO2011005861A17 Jul 201013 Jan 2011Alnylam Pharmaceuticals, Inc.Oligonucleotide end caps
WO2011017521A25 Aug 201010 Feb 2011Isis Pharmaceuticals, Inc.Bicyclic cyclohexose nucleic acid analogs
WO2011051333A127 Oct 20105 May 2011Novartis AgUniversal fibronectin type iii bottom-side binding domain libraries
WO2011056872A23 Nov 201012 May 2011Gen9, Inc.Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
WO2011085102A16 Jan 201114 Jul 2011Isis Pharmaceuticals, Inc.Base modified bicyclic nucleosides and oligomeric compounds prepared therefrom
WO2011094580A228 Jan 20114 Aug 2011Alnylam Pharmaceuticals, Inc.Chelated copper for use in the preparation of conjugated oligonucleotides
WO2011115818A110 Mar 201122 Sep 2011Isis Pharmaceuticals, Inc.5'-substituted bicyclic nucleosides and oligomeric compounds prepared therefrom
WO2011139695A226 Apr 201110 Nov 2011Isis Pharmaceuticals, Inc.Modified 5' diphosphate nucleosides and oligomeric compounds prepared therefrom
WO2011139699A226 Apr 201110 Nov 2011Isis Pharmaceuticals, Inc.5' modified nucleosides and oligomeric compounds prepared therefrom
WO2011139702A226 Apr 201110 Nov 2011Isis Pharmaceuticals, Inc.Modified nucleosides and oligomeric compounds prepared therefrom
WO2011139911A229 Apr 201110 Nov 2011Isis Pharmaceuticals, Inc.Lipid formulated single stranded rna
WO2011163121A120 Jun 201129 Dec 2011Alnylam Pharmaceuticals, Inc.Multifunctional copolymers for nucleic acid delivery
WO2012016245A21 Aug 20112 Feb 2012Novartis AgFibronectin cradle molecules and libraries thereof
WO2012064975A110 Nov 201118 May 2012Gen9, Inc.Protein arrays and methods of using and making the same
WO2012092053A121 Dec 20115 Jul 2012Eli Lilly And CompanyBovine viral diarrhea virus type 1b vaccine compositions and methods
WO2014063051A218 Oct 201324 Apr 2014Idexx Laboratories, Inc.Nucleic acid amplification controls and kits and methods of use thereof
WO2014072832A217 Oct 201315 May 2014Oslo Universitetstssykehus HfBiomarkers for cervical cancer
Classifications
U.S. Classification536/25.34, 536/26.71, 536/26.5, 987/189
International ClassificationC07H21/00, C07H21/04, C07H19/067, C07H19/20, C07H19/10, C07F9/26, C07H19/073
Cooperative ClassificationC07H21/00, C07F9/26
European ClassificationC07H21/00, C07F9/26
Legal Events
DateCodeEventDescription
19 Apr 1993ASAssignment
Owner name: MILLIPORE CORPORATION, MASSACHUSETTS
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:BIOSYNTECH GMBH;REEL/FRAME:006495/0626
Effective date: 19880516
2 Aug 1991FPAYFee payment
Year of fee payment: 4
27 Mar 1990RFReissue application filed
Effective date: 19900216
2 Jul 1987ASAssignment
Owner name: BIOSYNTECH GMBH, STRESEMANNSTRASSE 268-80, 2000 HA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:KOSTER, HUBERT;REEL/FRAME:004734/0865
Effective date: 19870626